acetate I. Glenn Sipes Department of Pharmacology, University of - - PowerPoint PPT Presentation

The in vitro hydrolysis of isoeugenyl acetate and eugenyl acetate

• I. Glenn Sipes

Department of Pharmacology, University of Arizona, Tucson, AZ

Determine the extent of hydrolysis for isoeugenyl acetate and eugenyl acetate in various biological matrices

Objective

ester

M.W 206.24

alcohol

M.W 164.20

O O R O O OH R

IA, R= EA, R=

Hydrolytic Reaction

Specific Aims

• Determine if skin, blood, and liver possess hydrolytic

activity towards isoeugenyl acetate and eugenyl acetate

• Establish stoichiometric relationship for the

disappearance of the esters and the formation of the alcohols over time

• Determine kinetic constants for the hydrolytic conversion

to the corresponding alcohols in the respective tissues.

• Assess the integrated metabolism of the esters in

monolayers of SD rat hepatocytes, an intact cellular system

Incubate 37oC sample prep

HPLC

• Reversed-phase alpha

bond C18 column

• Mobile phase: acidified

water and acetonitrile

• Monitoring

wavelengths of 254 nm and 265 nm Male SD rat IA or EA ~500 M

Blood Skin

Hepatocyte Isolation Subcellular Fractionation

Liver

Isolation and Treatment

• A. 0.5 min
• B. 5 min
• C. 20 min

min 10 12 14 16 18 20 22 24 26 28 mAU 50 100 150 200 250 300 350 DAD1 B, Sig=265,4 Ref=550,50 (CS010903\008-0901.D) min 10 12 14 16 18 20 22 24 26 28 mAU 50 100 150 200 250 300 350 DAD1 B, Sig=265,4 Ref=550,50 (CS010903\013-1501.D) min 10 12 14 16 18 20 22 24 26 28 mAU 50 100 150 200 250 300 350 400 DAD1 B, Sig=265,4 Ref=550,50 (CS010903\015-1701.D)

eugenol eugenyl acetate

Hydrolysis of EA by rat hepatic microsomes - (HPLC Profile)

Ester Hydrolysis in hepatic microsomal preparations

• Isoeugenyl-acetate concentration : 532 nmol/ml, (0.11 mg/ml) N=3
• Human female hepatic tissue also hydrolyzed IA to isoeugenol within 30 min
• Hydrolytic activity towards eugenyl-acetate is comparable for the respective

tissues

• A. Human Male
• B. SD Male Rat

10 20 30 100 200 300 400 500 600

Incubation Time (min) Concentration (nmol/ml)

10 20 30 100 200 300 400 500 600

Incubation Time (min) Concentration (nmol/ml)

Ester Hydrolysis in human blood and plasma preparations

• Isoeugenyl-acetate concentration : 488 nmol/ml, (0.10 mg/ml) N=2
• Blood and plasma from male SD rats also hydrolyzed both IA and EA.
• Studies indicate increased carboxylesterase activity in plasma preparations
• A. Blood:PBS (1:3)
• B. Plasma:PBS (1:10)

10 20 30 40 50 60 70 100 200 300 400 500

Incubation Time (min) Concentration (nmol/ml)

10 20 30 40 100 200 300 400 500 600

Incubation Time (min) Concentration (nmol/ml)

Hydrolysis of IA by rat skin microsomes - (HPLC Profile)

• A. 1 min
• B. 20 min
• C. 60 min

min 10 12 14 16 18 20 22 24 26 28 mAU 10 20 30 40 50 60 70 80 DAD1 B, Sig=265,4 Ref=550,50 (DC092503\012-1001.D) min 10 12 14 16 18 20 22 24 26 28 mAU 20 40 60 80 100 120 DAD1 B, Sig=265,4 Ref=550,50 (DC092503\018-1601.D) min 10 12 14 16 18 20 22 24 26 28 mAU 20 40 60 80 100 120 DAD1 B, Sig=265,4 Ref=550,50 (DC092503\020-1801.D)

isoeugenol isoeugenyl acetate

Ester Hydrolysis by rat skin microsomes and cytosol

• Isoeugenyl-acetate concentration : 488 nmol/ml, (0.10 mg/ml) N=4
• Microsomal Incubations consisted of 0.0375 mg/ml protein
• Cytosol Incubation consisted of 0.1 mg/ml protein
• Studies with eugenyl-acetate are comparable
• A. Microsomes
• A. Cytosol

10 20 30 100 200 300 400 500

Incubation Time (min) Concentration (nmol/ml)

10 20 30 100 200 300 400

Incubation Time (min) Concentration (nmol/ml)

Parameters for Kinetic Analysis

• Formation of isoeugenol and eugenol was measured in skin and hepatic

tissue preparations

• Skin Protein Concentrations: 0.0375 (M) & 0.1 mg/ml (C)
• Hepatic Protein Concentrations: 0.0125 (M) & 0.1 mg/ml (S-9)
• Dosing Range: IA [ 5 to 495 M], EA [39 to 970 M]
• Duration of incubation: skin (15 min) and hepatic (3 min)
• Kinetic Data was normalized to nmol / min / mg of protein

Concentration Dependent Ester Hydrolysis

• Formation of eugenol by male SD rat skin microsomes (left) N=2
• Formation of isoeugenol by human male hepatic microsomes (right)

N=3

250 500 750 1000 1250 200 400 600 800

M of Eugenyl-acetate

Rate (nmol/min/mg of protein)

100 200 300 400 500 600 1000 2000 3000 4000

M of Isoeugenyl-acetate

Rate (nmol/min/mg of protein)

Subcellular Fraction

CLint

(ml/min)

Vmax

(nmol/min/mg protein) IA EA

Km

(M) IA EA

Kinetic Constants for Alcohol Formation

• CLint = intrinsic metabolic clearance (Vmax / Km)
• Note the similarities between human and rat hepatic microsomes

with respect to the intrinsic metabolic clearance of EA and IA

Rat Hepatic S-9 Rat Hepatic Microsomes Human Male Microsomes Human Female Microsomes Rat Skin Microsomes Rat Skin Cytosol 638 60 3829 3656 2748 505 52 3822 3795 3072 97 190 223 110 173 91 97 72 89 51 52 137

IA EA

2.9 0.4 39.6 41.3 52.9 2.7 0.5 42.0 52.5 60.1 0.7 114 216 0.5

Integrated Metabolism of the esters in an intact cellular system

A. B.

• Isoeugenyl-acetate (A) concentration: 538 nmol/ml (0.11 mg/ml)
• Eugenyl-acetate (B) concentration: 483 nmol/ml (0.10 mg/ml)
• Within approximately 20 minutes hydrolytic conversion was complete

5 10 15 20 25 100 200 300 400 500 600

Incubation Time (min) Concentration (nmol/ml)

5 10 15 20 25 100 200 300 400 500 600

Incubation Time (min) Concentration (nmol/ml)

Summary

• IA and EA are rapidly hydrolyzed by tissue esterases to the

corresponding alcohols, isoeugenol and eugenol.

• Stoichiometric conversion to the alcohols was observed in

hepatic, blood, and skin preparations when incubated with IA and EA.

• When the localization of esterases was evaluated, the most

extensive activity was observed in the hepatic microsomal fraction.

• Human hepatic and blood preparations displayed similar

rates of hydrolysis to that of rodents when incubated under the same conditions.

Conclusions

• Toxicological data presently available for isoeugenol and

eugenol can be used for the safety assessment of the esters

• Animal data can be extrapolated to humans for rational

safety evaluation for the use of esters in fragrance and food products

• Absorption of IA and EA into the systemic circulation would

be minimal due to hydrolytic activity in skin, blood as well as the liver

• Slow rate of hydrolysis of esters in skin correlates

with their decreased sensitization potential

NOELs based on HRIPT

Compound NOEL

Isoeugenyl-acetate isoeugenol eugenyl-acetate eugenol 2 % 0.2-0.5 % 8 % ~ 5 %

OH OCH3 OH OCH3 O OCH3 O O

Conjugation Conjugation p-quinone methide

• -quinone

Hydrolysis Demethylation / Oxidation Oxidation

Potential reaction pathways for skin sensitization

IA EA

Acknowledgements

• Lab Members:
• David Castro
• Cathy Sweet
• Bob Kuester
• Research Associate:
• Aniko Solyom